Clinical Virology Manual, Fifth Edition

Affiliations: 1: Professor in the Department of Pathology, Director of the Clinical Microbiology Laboratory at the University of Texas Medical Branch (UTMB) at Galveston.;
2: Professor in the Microbiology Department and Chair of the Department of Biomedical Sciences at the University of South Carolina School of Medicine;
3: Director of Research and Clinical Trials at TriCore Reference Laboratories;
4: Assistant Professor in the Departments of Pathology and Medicine, Division of Infectious Diseases and Geographic Medicine, at the Stanford University School of Medicine

The clinical virology field is rapidly evolving and, as a result, physicians and clinical laboratory virologists must have a reliable reference tool to aid in their ability to identify and diagnose viral infections to prevent future outbreaks.

In this completely revised edition of the Clinical Virology Manual, Editor in Chief, Michael Loeffelholz, along with Section Editors, Richard Hodinka, Benjamin Pinsky, and Stephen Young, have complied expert perspectives of a renowned team of clinical virology experts and divided these contributions into three sections to provide

the latest information on the diagnosis of viral infections, including ebola, HIV and Human papillomavirus

state of the art diagnostic technologies, including next-generation sequencing and nucleic acid amplification methods

taxonomy of clinically important viruses such as polyomaviruses and zoonotic viruses

This comprehensive reference also includes three appendices with vital information on reference virology laboratories at the Centers for Disease Control and Prevention, state and local public health laboratories, and international reference laboratories and laboratory systems.

Additionally, a new section “Diagnostic Best Practices,” which summarizes recommendations for diagnostic testing, and cites evidence-based guidelines, is included in each viral pathogens chapter.

Clinical Virology Manual, Fifth Edition serves as a reference source to healthcare professionals and laboratorians in providing clinical and technical information regarding viral diseases and the diagnosis of viral infections.

General Topics in Clinical Virology

Viruses are a complex and diverse group of organisms that may have incredibly diverse and ancient origins. Their interaction with humans not only involves disease processes, but also evolutionary pressures that shape viral characteristics. Viral taxonomy, classification, and characterization is not a simple academic exercise but practically improves our ability to diagnose, track, and compare viruses of medical importance and develop a better understanding of pathophysiologic processes. Over the last 5 years, there have been significant changes in the proper names of some commonly identified viruses of medical importance, relationships between these medically relevant viruses, technologic tools, as well as websites and bioinformatics tools. Changes, including what constitutes the definition of a viral species, have already had an impact on how viruses are characterized and classified. The expanded utilization of whole genome sequence analysis and metagenomic approaches has increased the amount of biological information available to the scientific community for virus characterization and categorization. With these newer molecular approaches for virus identification and characterization, as well as enhanced bioinformatics approaches, viral classification is as dynamic and challenging as ever, requiring continuous monitoring, reassessment, and updating to achieve a rational taxonomic framework.

The clinical virology laboratory provides important and often critical information to the health care provider in order to support the diagnosis or monitoring of viral disease for the patient. Testing results will often serve as a guide for optimal treatment of the disease, contribute to infection control and prevention of a hospitalized patient or offer insight into the prognosis for the disease. Therefore, the quality of the virology laboratory testing has to be highly accurate and offered in a timely fashion in order to achieve optimal patient management. A well-structured and ongoing quality assurance (QA) program will provide the framework for maintaining accuracy in all phases of the testing process. These phases include the preanalytical, analytical, and postanalytical stages of the testing. However, no process is perfect, and every QA program should include a surveillance component that continuously identifies and corrects any weakness in the system. This corrective action should also be followed by preventative action in order to eliminate weaknesses and improve the entire QA program.

Clinical laboratories have come a long way since the 1900s when concerns were raised that they were too expensive and testing was too time consuming to be of practical use (1). In spite of those objections, laboratories have become the cornerstone of medical decision making. The beginning of laboratory regulation can be traced to the 1940s when Sunderman and Belk published findings from a voluntary survey of proficiency testing of regional laboratories that showed significant variation in laboratory performance (2). At around the same time, the College of American Pathology (CAP) was established, and one of its first functions was the initiation of national proficiency surveys in 1947 and 1948. The results further confirmed the need for standardization and regulation of clinical laboratories (3). In subsequent years, participation in proficiency surveys became standard practice among large hospital and reference laboratories (4). In 1967, Congress passed the Clinical Laboratory Improvement Amendment of 1967 (CLIA 67), which mandated certain minimum performance standards for reference laboratories involved in interstate commerce (4, 5). Similar regulation for hospital laboratories that were funded by Medicare followed in 1968 (6). The CLIA 67 regulations mandated that laboratories participate in “state approved or state operated proficiency testing programs” (5).

This chapter outlines the requirements for a safe environment in the clinical virology laboratory. This begins with development of a culture of safety which identifies the risks, develops a system to mitigate these risks, and encourages ongoing evaluation of the environment and continuous risk reduction. Chemical and fire safety, and decontamination and waste disposal, in addition to biosafety, are important components of an overall safety program. Classification of organisms by risk group and the corresponding biosafety containment levels are described. Routine work practices in clinical virology are identified along with recommended safe practices.

Planning and executing the design of a clinical laboratory is a unique and challenging task. This process not only involves coordinating how clinical samples will be received and tested, but also how efficiency can be maximized, safety ensured, and flexibility maintained. The design of a modern-day clinical virology laboratory can be especially challenging, given the continued application of traditional viral cell culture in some laboratories and the increasing use of molecular techniques (e.g., real-time PCR) for the diagnosis of viral infections. If there is one truth regarding laboratory design, it is that there is no “one size fits all” approach. Before a laboratory can begin to discuss the ideal approach for virologic testing at its institution, a number of important issues must be considered. These issues include the space that is (or will be) available, the number of laboratory staff that will occupy the space, the testing that will be performed, and the patient population from whom samples will be collected and submitted for testing. Furthermore, important decisions need to be made by laboratory and institutional leadership regarding whether testing should be performed in a centralized (i.e., consolidated) laboratory, a decentralized (i.e., specialized) laboratory, or a combination of the two. Another important consideration, driven by the increasing development of rapid, sample-to-answer molecular devices, centers around whether testing should be performed “near the patient” or at the “point of care.” Addressing each of these important issues is outside the scope of this chapter. However, key components of laboratory design, especially as they pertain to clinical virology, will be discussed to provide laboratory professionals with a foundation and guide to help ensure that test results are accurate, laboratory staff are safe, and future growth can be accommodated.

Laboratory Procedures for Detecting Viruses

Proper specimen collection is essential for interpreting test results because of the wide range of viruses, the complexity of virus–host interactions, and changes in testing methodology. Some viruses are part of our normal flora and cause no symptoms. Others, such as human herpes virus 6 (HHV-6), can integrate into germ line cells and be transmitted vertically to children (1, 2). Chronic, suppressed, or latent viruses like human immunodeficiency virus (HIV) or BK virus (BKV) can be reactivated to cause transient, low-level viremia that may or may not need clinical attention (3, 4). Asymptomatic shedding can also result in detection of virions for long periods of time (5–7). In order to recognize and interpret infectious episodes, it is crucial for diagnostic laboratories to receive appropriate specimens for viral testing.

Viruses are obligate intracellular parasites and thus are propagated using living cells in the form of cultured cells, embryonated hen's eggs, or laboratory animals. Culture has long been considered the “gold standard” for viral diagnosis because it secures an isolate for further analysis, is more “open-minded” than methods that target single agents, and allows the unexpected or even novel agent to be recovered. In practice, use of specialized cell culture systems, embryonated eggs, and laboratory animals is confined to research or major public health reference laboratories, with cell cultures in monolayers the sole isolation system utilized in routine diagnostic laboratories. The past two decades have seen conventional cell culture methods supplemented or even replaced by more rapid and targeted cell culture methods. Rapid culture methods can be performed by less experienced personnel, with less labor, and with results reported within 1 to 5 days of inoculation.

Laboratorians continually seek methodologies that yield accurate results in a timely fashion, are cost effective, and require less technical expertise. Diagnosis of viral infections via viral antigen detection methods such as immunofluorescence (FA), immunochromatography (lateral flow) (IC), and enzyme immunoassays (EIA) offer many of these attractive features and are useful for direct detection of viral antigens in an array of clinical specimens and for identification of cultivated viruses. Whether the detection method is FA, rapid IC, or EIA, detection of antigens of the common respiratory viruses (i.e., adenovirus; influenza virus [Flu] A and B; parainfluenza virus [PIV] −1, −2, and −3 and respiratory syncytial virus [RSV]), has been shown to be more useful in patient management than either traditional virus isolation (1, 2, 3) or viral detection in rapid culture using centrifugation-enhanced inoculation (4). There is considerable variability in the sensitivity, specificity, technical considerations, and turnaround time among the various methods, and each method may perform differently depending on the viral target. This chapter deals with principles of FA, IC, and EIA and their contemporary applications in viral antigen detection.

For communicable diseases, clinical management, and public health response, it is often important to know the body's immune response following exposure and infection with pathogens. Although humoral and cell-mediated immunity both play roles in the body's specific immunity against viral pathogens, testing antibody response for humoral immunity is much more common and is also easier in clinical virology laboratories than testing for cell-mediated immunity because of the convenience of antibody serological testing methods. There is a long history of using various serologic methods in clinical virology laboratories for antibody detection. Some methods, such as complement fixation test and immunodiffusion test, have been gradually phased out and replaced by faster and less laborious methods (1, 2). In this chapter, we will focus on antibody detection methods used in clinical virology laboratories: neutralization, hemagglutination inhibition, indirect immunofluorescence, enzyme immunoassay, and Western blot.

Nucleic acid (NA) extraction is a critical step used in molecular biology and molecular diagnostics (1–5). Successful extraction of NA depends on the quantitative recovery of pure molecules of RNA and DNA in an undegraded form. Salts, for example, are common impurities in NA samples, and it is important that they are removed from NA before any downstream processes and analyses can be performed (1). Therefore, single or multiple separation and/or purification steps are needed to desalt the sample containing the NA. The process of extraction and purification of NA also removes a variety of inhibitors of downstream NA amplification procedures. The first step of NA extraction and purification involves cell lysis to liberate NA from cell nuclei or pathogens. Effective NA extraction methods include reagents that inactivate nucleases (DNase and RNase) to preserve the NA in an intact state. The final steps involve separation and recovery of the NA free of cellular debris, proteins, and various potential inhibitors of downstream assays (1–5).

Nucleic acid detection methods have been rapidly evolving and play an important role in viral detection and quantification. One technology which emerged in the 1990s, polymerase chain reaction, better known as PCR, has established itself as a primary tool for molecular biology. In fact, this technology has been so widely adopted in the clinical virology laboratory it has, in many cases, completely replaced culture. At its core, PCR is a straightforward chemical reaction whereby one strand of template DNA is exponentially amplified. This chapter will give an overview of different PCR technologies available, as well as the strength and weaknesses of each. For a more in-depth review of their applications in the clinical laboratory, the reader is directed to the appropriate chapter(s) elsewhere in this text.

In clinical virology, molecular diagnostics based on the direct detection of specific genetic material in a specimen through nucleic acid testing (NAT) has largely replaced antigen testing by immunoassays and has become the leading technology. Molecular test systems are more specific and sensitive. They are able to detect the presence of a pathogen earlier than an antigen immunoassay and are thus mainly used for diagnosing and screening patients for numerous viral diseases today.

Diagnostic information describing the actual or relative density of specific viral nucleic acids in a human blood sample is most easily ascertained using quantitative molecular methods (aka viral loads), whose historical use spans more than two decades. A common function of quantitative viral load testing is to support clinical strategies and practices that monitor patients for human immunodeficiency virus (HIV) and most of the hepatitis viruses. For transplant patients, viral loads are monitored to detect numerous viruses classified in the herpes virus group, including cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV-6), among others. It seems that each year, there is a new application or interpretation for results of viral load assays. Bacterial load assays are uncommon; however, there is some discussion of its use for certain conditions.

Signal amplification methods were initially designed as an alternative to the target amplification technologies such as polymerase chain reaction (PCR) so as to minimize the possibility of contamination by target amplification products. Unlike target amplification, signal amplification methods (as defined herein) do not rely on enzymes for the amplification. Probe-based amplification techniques such as cleavage-based amplification (36) and rolling-circle amplification (1) rely on enzymes, and will not be covered in this chapter. Signal amplification increases or amplifies the signal generated from the probe molecule hybridized to the target nucleic acid sequence. The advantages of signal amplification methods include specific detection, dynamic range, ease-of-use, and reproducibility. To date these methods have met the challenge from advanced or automated target amplification methods such as real time PCR. Signal amplification technologies include hybrid capture (HC) and branched DNA (bDNA) assays (32, 33). The HC method was developed and marketed initially by Digene Corporation (Gaithersburg, MD) which was acquired by Qiagen (Valencia, CA) in 2007. The bDNA method was initially developed by Chiron (Emeryville, CA), marketed by Bayer Diagnostics (Emeryville, CA) which diagnostic division was acquired by Siemens (Tarrytown, NY) in 2006. Due to the growing demand for quick time to detection, automation, and multiplexing, the popularity of commercial signal amplification methods has declined in clinical virology laboratories.

Recent advances in sequencing technology, coupled with the relatively small genomes of viruses, make routine sequencing of entire genomes in clinical and public health settings increasingly feasible. The first two widely adopted DNA sequencing methodologies described, the chemical cleavage method of Maxam and Gilbert (1) and the chain termination method of Sanger et al., were both published in the 1970s (2, 3). Coincidental to the subject of this chapter, the first full genome to be sequenced by Sanger was that of a virus, albeit a bacteriophage, PhiX 174 (4). The Sanger method proved to be the more durable sequencing technology and, especially after the process was automated in 1996, was the most widely used method for DNA sequencing for more than a decade. Beginning in 2005, however, advances in sequencing technology, the so-called next generation sequencing (NGS) methodologies, resulted in a dramatic increase in the amount of sequence that can be generated and a concomitant dramatic decrease in the cost of sequencing. These factors have led to the widespread implementation of NGS in place of the Sanger method for typical sequencing applications and also for some novel purposes, for example, replacing microarrays to study gene expression. The increased use of NGS technologies has, not surprisingly, resulted in a rapid increase in the number of sequences submitted to the National Center for Biotechnology Information's (NCBI) Genbank database. In particular, the number of viral sequences submitted since 2012 has increased 22.9% as measured in nucleotide base pairs (5), and the number of publications based on NGS is increasing at an impressively rapid pace (6).

Where the use of antibiotics goes back to the discovery of penicillin by Alexander Fleming in 1928, application of antiviral treatment was not achieved until the early 1960s with the use of the nucleoside analogue idoxuridine for treatment of herpetic keratitis (1) and methisazone for treatment of smallpox, variola, and cowpox. The first major advances in antiviral treatment were obtained for herpes viruses with the discovery of another nucleoside analogue, acyclovir, by the Burroughs Wellcome Company in the early 1980s. Despite these advances, the real wave of antiviral drug discovery was the result of the HIV epidemic, and started with the development of azidothymidine (AZT) as the first antiretroviral for AIDS patients (2). Nowadays a wide spectrum of antiviral agents are used for a variety of infections. However, similarly to bacteria, development of resistance is an important complication when using antiviral agents. In addition, many viral pathogens have an RNA genome and use RNA polymerases that lack proofread activity for their replication. Therefore, mutations will be introduced into the viral genome in every replication cycle and as a consequence an altered susceptibility or even resistance to an antiviral agent may develop. These resistant variants are easily selected under pressure of an antiviral treatment as shown by the rapid development of resistance when using AZT (3, 4). The viral reverse transcriptase incorporates this thymidine analogue into the viral genome, inhibiting proper replication. Resistant viruses rapidly evolve by acquiring resistance-associated mutations (RAM) in the gene encoding for the enzyme. With HIV being a retrovirus, these mutations are also incorporated in the host DNA and therefore the resistant viral genome is stored in the DNA of the patient and will emerge upon reintroducing the drug.

A point-of-care test (POCT) may be defined as an analytical or diagnostic test that is performed at the bedside or in a near-patient setting, a location distinct from a typical hospital laboratory (1). POCTs are technically less complex than traditional laboratory tests and therefore can be performed by health care professionals or nonmedical personnel. Some laboratories may also use POCTs as a rapid alternative to conventional methods or in facilities where complex testing is limited. Key features of POCTs are listed in Table 1. To be of value, POCTs should afford rapid results and have a high degree of sensitivity and specificity compared to more complex traditional testing performed in a laboratory. With reliable POCT results in hand, providers can make patient management decisions that improve outcomes for the patient or hospital. In some instances the impact may also be a more cost-effective solution compared to laboratory-based testing. POCTs should be simple to perform and interpret by nonlaboratory personnel using uncomplicated instrumentation, contain internal controls to ensure validity of results, have temperature-stable components that allow easy and prolonged storage, and be relatively inexpensive.

This is a very exciting time for the field of clinical diagnostics. In recent years, there has been an explosion in new methodologies and instrumentation to diagnose infection, with a focus on optimization of therapy and antimicrobial stewardship. The result is new technology that improves the diagnosis, characterization, and monitoring of viral infections. These technologies are changing almost every aspect of the laboratory workflow. Most of these new methods achieve marked improvements in turnaround time compared to conventional methods, but this often comes at an increased cost, might be technically demanding, and may require specialized equipment. It is unclear how many of these emerging technologies will rise to widespread, routine clinical use. There are a number of challenges that precede widespread adoption, including regulatory approval and demonstration of adequate analytical performance characteristics. This chapter highlights and summarizes some of the emerging technologies for the diagnosis of viral infections, including digital PCR, next-generation sequencing methods, mass spectrometry, surface plasmon resonance assays, and novel approaches to point-of-care diagnostics. The strengths and limitations of each methodology, as well as potential clinical diagnostic applications, will be described.

Viral Pathogens

Respiratory tract illnesses are one of the most common health conditions affecting humans. Most of these illnesses are caused or triggered by viruses. Clinical presentations and severity range from mild and self-limited upper respiratory tract illnesses (URTI) to serious or fatal lower respiratory tract disease. Respiratory viruses exert considerable pressure on health care systems, are significant drivers of antibiotic overuse, and contribute significantly to loss of productivity. Accurate identification of these infections and appropriate care of affected patients are therefore priorities for health care systems.

Human enteroviruses (EV) are members of the Enterovirus genus of the family Picornaviridae and are among the most common human viral infections. Their discovery had implications for all of virology because they indicated, first, that poliovirus (PV) grew in various tissue culture cells that did not correspond to the tissues infected during the human disease and, second, that PV destroyed cells with a specific cytopathic effect (CPE). The infectious virus is relatively resistant to many common laboratory disinfectants, including 70% ethanol, isopropanol, dilute Lysol, and quaternary ammonium compounds. Poliomyelitis should be considered in all cases of pure motor paralysis and is usually associated with a normal or slightly elevated value for protein, normal sugar value, and moderate mononuclear pleocytosis in cerebrospinal fluid (CSF). Real-time PCR is the primary test used to detect, even with very small amounts of clinical specimens, such as CSF. All important information about a virus could potentially be obtained directly by PCR in conjunction with nucleic acid sequencing if all the molecular correlates of viral phenotypic determinants were understood. The most common molecular typing system is based on reverse transcription (RT)-PCR and nucleotide sequencing of a portion of the genomic region encoding VP1. Another group of pathogenic picornaviruses, the human parechoviruses (genus Parechovirus), cause a similar array of illnesses as the EV, and they are detected clinically by distinct molecular assays that are analogous to those used for the EV.

Measles virus is a single stranded, nonsegmented, negative sense RNA virus and the prototypic member of the Morbillivirus genus of the Paramyxovirnae subfamily of the Paramyxoviridae. The standard viral genome is 15,894 nucleotides in length and contains six genes and encodes eight proteins which include nucleoprotein (N), phosphoproteins (P) C and V, and matrix (M), fusion (F), hemagglutinin (H), and polymerase (L) proteins. The measles virion is spherical with a diameter ranging from 120 to 250 nm. The virus buds from the plasma membranes of infected cells and has an envelope composed of glycoproteins, the H and F proteins, and lipids. The H and F proteins appear as short surface projections and are responsible for receptor binding and virus entry into susceptible cells (1). Three cell surface receptors for wild-type measles virus have been identified and all interact with the H glycoprotein (2). The M protein is positioned under the virion envelope and anchors the nucleocapsids to the budding sites at the plasma membrane. Unlike the H and F surface proteins, the M is neither glycosylated nor transmembranous. The envelope encloses an elongated helical nucleocapsid in which protein units are spirally arranged around the nucleic acid. The nucleoprotein (N), phosphoprotein, and large polymerase protein, in conjunction with the virion negative strand RNA, comprise the ribonucleoprotein complex, the replicating, and transcriptional unit of measles virus (1, 3). Although measles virus has only a single serotype, it can be subdivided into 24 distinct genotypes based on the sequence variability of the last 450 nucleotides of the N gene. These sequences can vary by up to 12% among the genotypes and form the basis of the molecular epidemiology applied to tracking of transmission pathways, monitoring control measures, and distinguishing wild-type viruses from vaccine strains of measles (4, 5).

Since the demonstration by Albert Kapikian and colleagues that a virus was the etiological agent of Norwalk gastroenteritis (1), multiple viruses have been recognized as significant causes of gastroenteritis in children and adults. Gastrointestinal viruses have been implicated in food and waterborne outbreaks of gastroenteritis, seasonal disease, and outbreaks in all age groups in both the developed and developing worlds. The major gastrointestinal viruses causing acute gastroenteritis are found in several virus families (Table 1) and will be discussed in greater detail in the following text.

Viral hepatitis refers to inflammation of the liver caused by several viral agents, including hepatitis A (HAV), B (HBV), C (HCV), D (HDV), and E (HEV) viruses. Generally, HAV and HEV are associated with acute, self-limited infection; however, severe and protracted disease can develop. These nonenveloped viruses have independently evolved interesting mechanisms aimed at evading the immune response and guaranteeing survival in the host. These viruses are phylogenetically unrelated but share several similarities. The genomes from both viruses are single-stranded, positive-polarity RNA. The viruses primarily infect the liver, cause acute infection, and are shed in feces. Moreover, the clinical manifestations from HAV and HEV infection are undistinguishable and require specific laboratory tests for each virus. Despite the similarities exhibited by these viruses, differences in the epidemiology of the respective diseases result in distinctive distribution patterns worldwide.

Hepatitis was first described in the fifth century B.C. The earliest known outbreak of hepatitis occurred in Bremen, Germany, in 1883 among shipyard workers who received a smallpox vaccine stabilized with human serum. In 1950, viral hepatitis was referred to as either infectious hepatitis (hepatitis A) or serum hepatitis (hepatitis B) based on the epidemiologic characteristics of the diseases (1). This terminology was adapted by the World Health Organization (WHO) in 1973.

Hepatitis C virus (HCV) is a single-stranded RNA virus that was not known as a causative agent of acute and chronic hepatitis until 1989. The development of blood transfusion technology in the mid-20th century led to its rapid spread into new populations with enormous disease burden due to cirrhosis and hepatocellular carcinoma (1, 2). Its discovery by reverse molecular genetics is one of the major achievements of modern medicine and a model for the discovery of many other pathogens during the past 25 years (3). Unlike hepatitis B virus (HBV) and human immunodeficiency virus (HIV), which establish permanent chronic infection, HCV is spontaneously cleared in some individuals and is also amenable to therapeutic cure. With advances in drug therapies, all HCV infections are potentially curable. The development of technologies for viral discovery and clinical detection and monitoring, as well as new classes of antiviral therapy, are important components of the HCV legacy.

The herpesviruses are classified in the family Herpesviridae based on the characteristic large linear double-stranded DNA genome packaged within an enveloped icosahedral capsid. Members of this family have been identified in almost all animal species, and the specific herpesviruses are usually restricted to a single species. Nine human herpesviruses have been described: HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (CMV), human herpesvirus 6A (HHV-6A), human herpesvirus 6B (HHV-6B), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8). All these viruses are structurally similar and encode a large number of biosynthetic enzymes involved in the synthesis of viral DNA as well as structure proteins that compose the capsid and tegument, as well as envelope glycoproteins that are required for infection.

Cytomegalovirus (CMV) is one of 8 known herpesviruses to infect humans. Classified as a β-herpesvirus, along with human herpesvirus-6 and human herpesvirus-7, CMV is a large virus that was first isolated in humans from salivary glands in the 1950s (1). Since its discovery, CMV has been linked to a multitude of clinical syndromes in humans and is possibly the most important virus of clinical consequence among immunocompromised patients and pregnant women.

In 1964, three researchers, Michael Anthony Epstein, Bert Achong, and Yvonne Barr, published in The Lancet their discovery of what would later be known as Epstein-Barr virus (EBV) (1). Before this breakthrough, Epstein had been studying chicken tumor viruses at the Middlesex Hospital in London. In 1961, Epstein attended a lecture by Denis P. Burkitt, a British surgeon who had been stationed in Uganda, in which he detailed the relationship between Burkitt lymphoma and the geographical patterns of temperature, rainfall, and altitude (2). Suspicious of a viral etiology, Epstein spent the next few years attempting to isolate viral material from lymphoma biopsy samples taken from tumors of Ugandan children sent weekly by Burkitt to London. Despite a switch to tissue culture and assistance from Yvonne Barr and Bert Achong, isolation of a virus was unsuccessful. On December 5, 1963, the sample sent by Burkitt was delayed due to inclement weather, which fortuitously resulted in viable, free-floating, lymphoma cells that astonishingly grew in culture and demonstrated viral particles by electron microscopy (1). Though this initial finding was met with skepticism, mounting evidence over the following decades eventually resulted in the acceptance of EBV as the cause of Burkitt lymphoma, and the World Health Organization and the International Agency for Research and Cancer declared EBV as a group 1 carcinogen in the 1990s (2). Approximately 90% of people worldwide are carriers of latent EBV, and it is estimated that the virus causes more than 200,000 cancers each year, primarily B-cell neoplasms, which account for 1.5% of all cancers (2). There are two types of EBV, EBV-1 (A type) and EBV-2 (B type), that are distinguished by differences found primarily in their latent genes. EBV in America and Europe are much more likely to be EBV-1, whereas EBV found in Africa may be EBV-1 or EBV-2.

Human herpesvirus 6 (HHV-6A and -6B variants), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8) were the last three herpesviruses identified, from 1986 to 1994 (1–3). HHV-6 and HHV-7 are ubiquitous viruses showing >95% prevalence worldwide from early childhood, usually with asymptomatic primary infection. HHV-6 has been associated with many clinical conditions, but attempts to establish clear etiologic relationships with human disease have largely been confounded by the ubiquity of HHV-6 and the only relatively recent formal recognition of variants HHV-6A and HHV-6B. The most well-recognized clinical presentations of HHV-6 infections, roseola infantum and fever, occur as a result of primary infection mainly in otherwise healthy infants and young children. Primary infection in adults is uncommon. Severe syndromes typically result from HHV-6 reactivation in immunocompromised hosts. Most infections are caused by HHV-6B; disease attributed to HHV-6A is rare. Both of the HHV-6 variants and HHV-7 have been associated with disease in immunocompromised organ transplant recipients. HHV-8 stands out as being the only human herpesviruses with low prevalence in most of the world and is mainly known as the etiologic agent of Kaposi's sarcoma (KS). Like other herpesviruses, these viruses establish lifelong infections in their host and are maintained through a combination of latent (nonproductive) infections and intermittent or persistent lytic infections; consequently, they present diagnostic challenges.

Papillomaviruses are species specific; in humans they infect a number of sites such as the skin, mouth, anus, conjunctiva, and lower genital tracts of both males and females. The majority of infections, no matter the site, are typically asymptomatic and subclinical. Genital human papillomavirus (HPV) is the most common sexually transmitted disease. It has been established and accepted that oncogenic or high-risk HPV types are the main cause of cervical cancer in women and can cause other cancers such as vulvar, vaginal, penile, anal, and oropharyngeal cancer. Although persistent infection with a high-risk HPV type is necessary for the development of cervical cancer, many women will spontaneously clear the infection and are subsequently not at risk for developing cancer in the future. It is known that greater than 70% of cervical cancer cases are due to HPV types 16 and 18, and testing for HPV along with Pap smear testing is a widely accepted approach for cervical cancer screening. Several organizations have developed guidelines for cervical cancer screening in United States (US) including the United States Preventive Services Task Force (USPSTF), American Cancer Society (ACS), the American Society for Colposcopy and Cervical Pathology (ASCCP), and the American Society of Clinical Pathology (ASCP). Recent data suggest that HPV testing can be used as the primary screen for cervical cancer screening as algorithms continue to evolve around clinical patient management. Prevention of cervical cancer and other cancers is now feasible because of the availability of HPV vaccines.

The parvoviruses are a large group of DNA viruses capable of infecting a wide variety of both invertebrate and vertebrate hosts, including humans and their companion animals. Depending on the virus and the immune status of the host, human infection can range from overt disease to persistent asymptomatic infection. Current parvovirus taxonomy dates from 2004; however, in the intervening 10 years, as new viruses have been identified and more taxonomic data have become available, the current taxonomy is now considered outdated. A new taxonomic classification of the family Parvoviridae has therefore been proposed and is currently under review and likely to be instituted in the near future (1). The proposed taxonomy, based on the amino acid sequence of the NS1 and viral capsid proteins, maintains the family Parvoviridae and subfamily Parvovirinae for the vertebrate parvoviruses, but adds three new genera and a nomenclature change that impact the taxonomy of the human parvoviruses. A member of a proposed genus must now have >30% amino acid sequence identity to other members of the same genus but <30% identity to members of other genera (1). Criteria for inclusion in a viral species have also changed under the proposed taxonomy, with members of the same species having >85% amino acid sequence identity to other members of the same species and >15% amino acid diversity from members of other species (1). The proposed genera and the human viruses they contain are Bocaparvovirus: human bocaviruses 1–4 (HBoV1-4); Dependoparvovirus: human adeno-associated viruses (AAVs); Erythroparvovirus: human parvovirus B19-related viruses; Protoparvovirus: human bufaviruses; and Tetraparvovirus: human parvovirus 4–related viruses (Par4) (1).

Variola virus (VARV), a member of the Orthopoxvirus genus, caused one of the most feared illnesses of mankind, smallpox. In 1798, Edward Jenner described that milkmaids with evidence of prior infection with cowpox (caused by Orthopoxvirus Cowpox virus [CPXV]) were immune to infection with smallpox (VARV). Smallpox vaccines, derived from Orthopoxvirus Vaccinia virus (VACV), were subsequently used extensively for routine vaccination against VARV. Through an intensive vaccination campaign, coordinated by the World Health Organization (WHO), naturally occurring VARV infections were declared eradicated in 1980. These modalities are also of interest in recognition and control of emerging zoonotic orthopoxviruses (Monkeypox virus [MPXV], CPXV, and VACV).

Rabies is the prototype virus of the Lyssavirus genus of the order Mononegavirales, family Rhabdoviridae. The Mononegavirales are characterized by a nonsegmented, negative-stranded RNA genome, encapsulated tightly into a ribonucleocapsid structure. The Rhabdoviridae are classified as a group based on a similar conical or bullet-shaped appearance by electron microscopy. The host range for Rhabdoviridae is highly diversified, including plants, arthropods, fish, and mammals (1). Previously known simply as the rabies and rabies-related virus group, the genus Lyssavirus is presently composed of genotype 1, classical rabies virus, and 14 other genotypes that are closely related antigenically and genetically and produce a clinical disease indistinguishable from rabies (2). However, lyssaviruses are serologically distinct from other rhabdoviruses.

Arboviruses (arthropod-borne viruses) are a biologically defined category of viruses that almost exclusively have RNA genomes. Most of the 100 medically important arboviruses belong to five families: Togaviridae, Flaviviridae, Bunyaviridae, Rhabdoviridae, and Reoviridae. A single genus in the family Orthomyxoviridae (Thogotovirus) and a single DNA virus in the family Asfarviridae (African swine fever virus) are also members of the group. The arboviruses include approximately 40 serological groups based on antigenic cross-reactivity. These viruses are unique in that they generally require cycling between disparate hosts (i.e., vertebrates and hematophagous arthropod vectors). Mosquitoes and ticks are the most common invertebrate vectors, while less common vectors include biting midges and sandflies. Some viruses, such as vertebrate-only flaviviruses (Rio Bravo virus, for one) and insect-only alpha- and flaviviruses (Kamiti River virus, for one) are not transmitted between disparate hosts.

Zoonotic infections are infections of animals that can be transmitted to humans. There are more than 400 viruses with a zoonotic origin that can cause mild or severe clinical pathology in humans. This chapter will attempt to cover a handful of these viruses that have been especially relevant as human pathogens: arenaviruses, Ebola virus, Nipah virus, and Hantavirus. Arenaviruses consist of a number of species and collectively have a worldwide distribution. They cause mild to severe illness in humans. Filoviruses have a sylvatic epidemiology in Africa and can spill over into humans, where they usually cause severe illness with a high mortality rate. Filoviruses are efficiently spread person-to-person, as highlighted in the 2014 to 2015 urban epidemic in West Africa. Nipah virus has caused a relatively limited number of human cases in Southeast Asia. Nipah virus infection with central nervous system involvement is associated with high mortality. Hantaviruses have a worldwide distribution and cause renal and pulmonary syndromes in humans.

The human immunodeficiency viruses (HIV) and the human T-lymphotropic viruses (HTLV) originate from zoonotic transmission of ancestor retroviruses found in primates. HIV-1 and HIV-2 are the causative agents of AIDS; HTLV-1 causes adult T-cell leukemia/lymphoma and a broad spectrum of chronic inflammatory diseases, though only in few of those infected. The three principal questions in HIV diagnostics are (i) whether a person is HIV-infected and, if infected, (ii) with what exactly (viral properties) and (iii) how actively the virus is replicating (viral load). With regard to viral properties, identification of the virus type, i.e., HIV-1 or HIV-2, is essential for both the selection of a suitable viral load test for HIV RNA quantification in plasma and the decision with what to treat the patient. Answering these questions is important because current commercial viral load tests do not recognize HIV-2, and nonnucleoside reverse transcriptase inhibitors (NNRTI) are not effective against HIV-2 or group O viruses of HIV-1. Knowledge of preexisting viral mutations conferring resistance to antiretrovirals is another important point. As antiretroviral therapy is increasingly considered essential for all HIV infected persons, it is important to answer all relevant questions already at the timepoint of HIV diagnosis. Diagnosis of HTLV infection is performed accordingly, but with some important differences. HTLV never leads to viremia. Tests for viral RNA in plasma are thus useless, and all nucleic acid testing has to be performed on cells. The most important task is to differentiate between the pathogenic HTLV-1 and the virtually nonpathogenic HTLV-2.

The Chlamydiaceae are a family of small, metabolically dependent bacteria with a unique intracellular life cycle (1). As a result of the obligate intracellular growth of these organisms in eukaryotic cells, they are handled in ways more closely resembling the detection of viruses than bacteria. Thus, inclusion of these organisms in this manual is appropriate.

The human microbiome is the collection of hundreds of trillions of microorganisms and their genomes that colonize the human body. Recent advances in sequencing technology have allowed these communities and their function to be characterized and defined in detail. While the bacterial members of the microbiome have received the bulk of the attention, the viral members, the human virome, promise to be no less important. This chapter describes these recent advances in the methods for metagenomic studies, highlights key early findings, and looks ahead to the remaining challenges to implementing viral metagenomics and human virome techniques in the clinical setting for diagnostic purposes.

Host genetic variation in components of both specific and innate immune responses affects susceptibility to viral infections. Innate immunity provides the first line of defense, and the development of adaptive immunity is stimulated by innate responses. Pathogen recognition receptors (PRRs) initiate signaling pathways that result in the production of antiviral interferons and cytokines. Mutations or genetic variants (polymorphisms) have been recognized in several factors of innate immunity. Notably, human populations from distinct geographic areas have different frequencies of immune gene variants. The genetic susceptibility may vary from life-threatening manifestations of specific virus infections to a moderately increased frequency of nonsevere infections. Although the innate immunity is nonspecific by nature, the reactions are stereotypic for viral infections compared with bacterial infections. Even infections caused by specific viruses can be differentiated from each other based on the innate immune response. Host response pattern determination by expression analysis of a predefined set of genes is a novel strategy in the diagnosis of virus infections. Another strategy in differentiating viral and bacterial infections from each other could be the determination of a single marker, such as myxovirus resistance protein A (MxA), which is generally induced by viruses but not by bacteria. Host response analysis could also be used in monitoring infections and antiviral treatment, but applications for routine use are not yet available. Certain host gene variants correlate with the prognosis of infection. Currently, for instance, interleukin (IL) 28B genotyping is used to aid in hepatitis C treatment decisions.